The present invention relates to aminopeptidases and genes encoding them.
Koji molds are used for the preparation of soy sauce, miso (fermented soybean paste) and other natural seasonings containing protein hydrolysates. For example, soy sauce is produced in two steps, i. e. a step of preparing koji and fermentation step. The starting materials are hydrolyzed by an enzyme produced by a koji mold (a filamentous fungus belonging to the genus Aspergillus) in the step of preparing the koji mold. For improving the taste of the soy sauce, it is important to increase the amount of free amino acids in soy sauce in these steps.
Amino acids are produced from the starting protein by two steps. The first step is the release of peptides from proteins by proteases and the second step is the production of amino acids through hydrolysis of peptides, catalyzed by peptidases.
As for peptidases of koji mold, those derived from Aspergillus oryzae and Aspergillus sojae were reported (JP-Kokai No. 11-346777, DE 95-1952648, WO 9851163, WO 9628542, WO 9615504, WO 9851803 and WO 9814599). It is described therein that leucine aminopeptidase is particularly important in the preparation of soy sauce. However, it has not been reported that known leucine aminopeptidase is resistant to salt. As for the genes of leucine aminopeptidase of the genus Aspergillus, Kaifu et al. reported Aspergillus sojae (JP-Kokai No. 11-346777), but there has been no report on the salt-resistance of this enzyme.
As for the genus Bacillus, there is a reported of salt-resistant leucine aminopeptidase (Lee, G. D. et al., J. Appl. Microbiol. (1988), 85 (3)).
On the other hand, Asano et al. noted that storage proteins in soybean are hydrolyzed into amino acids in a very short period of time in the course of the germination thereof. They found peptidases (aminopeptidase GX capable of efficiently hydrolyzing acidic amino acid-containing peptides and leucine aminopeptidases) in soybean cotyledons and succeeded in effectively hydrolyzing soybean protein (JP-Kokai No. Hei 9-294583).
In view of the enzymologic properties of aminopeptidase GX of soybean, aminopeptidase GX was a new aminopeptidase that had never been reported. The presence of the aminopeptidase GX of soybean had not been known except in germinating soybean. Aminopeptidase GX of soybean has an activity of effectively releasing N-terminal acidic amino acids from peptides having acidic amino acids such as glutamic acid at the N-terminal thereof. Accordingly, it is possible to produce soy sauce having a high free glutamic acid content and an excellent taste, taking advantage of the effect of this enzyme.
Ninomiya et al. succeeded in producing a large amount of soybean aminopeptidase GX by a genetic recombination technique (JP-Kokai No. 2000-325090). However, it is difficult to use aminopeptidase GX of soybean produced by this method for the production of soy sauce because of the problems of GMO and costs.